CD73, CD90, CD105 and Cadherin-11 RT-PCR Screening for Mesenchymal Stem Cells from Cryopreserved Human Cord Tissue.

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs) are self-renewing, non-specialized cells used clinically in tissue regeneration and sourced from bone marrow, peripheral blood, umbilical cord blood and umbilical cord tissue (UCT). To demonstrate an alternative method for MSC detection, cryopreserved UCT and expanded MSC were screened for MSC markers CD73, CD90, CD105 and CDH-11 by RT-PCR. METHODS AND RESULTS: Human UCT were washed, sectioned, cryopreserved with 10% DMSO and stored in the vapor phase of liquid nitrogen. Fresh and frozen UCT samples were expanded for MSC. UCT and MSC were processed for RNA and screened for CD73, CD90, CD105 and CDH-11 mRNA by RT-PCR. CD73, CD90 and CD105 were detected by flow cytometry and CDH-11 was detected by Western blotting. Short and long-term frozen UCT shows a loss of mRNA expression for CD73 at 33.2±34.0%, CD90 at 6.2±8.2%, CD105 at 17.7±21.6% and CDH-11 at 30.1±26.7% but was not statistically significant to indicate the deterioration. Expanded MSCs from fresh UCT expressed 0.09±0.07-fold CD73, 0.17±0.11-fold CD90, 0.04±0.06-fold CD105 and 0.14±0.08-fold CDH-11. Expanded MSCs from frozen UCTs expressed 0.09±0.06-fold CD73, 0.13±0.06-fold CD90, 0.04±0.05-fold CD105 and 0.11±0.06-fold CDH-11 and confirmed by flow cytometry and Western blotting. CONCLUSION: CD73, CD90, CD105 and CDH-11 were detected by RT-PCR in cryopreserved UCT and MSC expansion. CDH-11 appears as a useful single target MSC marker for quick screening.

Progress toward curing HIV infection with hematopoietic cell transplantation.

HIV-1 infection afflicts more than 35 million people worldwide, according to 2014 estimates from the World Health Organization. For those individuals who have access to antiretroviral therapy, these drugs can effectively suppress, but not cure, HIV-1 infection. Indeed, the only documented case for an HIV/AIDS cure was a patient with HIV-1 and acute myeloid leukemia who received allogeneic hematopoietic cell transplantation (HCT) from a graft that carried the HIV-resistant CCR5-∆32/∆32 mutation. Other attempts to establish a cure for HIV/AIDS using HCT in patients with HIV-1 and malignancy have yielded mixed results, as encouraging evidence for virus eradication in a few cases has been offset by poor clinical outcomes due to the underlying cancer or other complications. Such clinical strategies have relied on HIV-resistant hematopoietic stem and progenitor cells that harbor the natural CCR5-∆32/∆32 mutation or that have been genetically modified for HIV-resistance. Nevertheless, HCT with HIV-resistant cord blood remains a promising option, particularly with inventories of CCR5-∆32/∆32 units or with genetically modified, human leukocyte antigen-matched cord blood.

Hematopoietic cell transplantation with cord blood for cure of HIV infections.

Hematopoietic cell transplantation (HCT) using CCR5-Δ32/Δ32 stem cells from an adult donor has resulted in the only known cure of human immunodeficiency virus (HIV) infection. However, it is not feasible to repeat this procedure except rarely because of the low incidence of the CCR5-Δ32 allele, the availability of only a small number of potential donors for most patients, and the need for a very close human leukocyte antigen (HLA) match between adult donors and recipients. In contrast, cord blood (CB) transplantations require significantly less stringent HLA matching. Therefore, our hypothesis is that cure of HIV infections by HCT can be accomplished much more readily using umbilical CB stem cells obtained from a modestly sized inventory of cryopreserved CCR5-Δ32/Δ32 CB units. To test this hypothesis, we developed a screening program for CB units and are developing an inventory of CCR5-Δ32/Δ32 cryopreserved units available for HCT. Three hundred such units are projected to provide for white pediatric patients a 73.6% probability of finding an adequately HLA matched unit with a cell dose of ≥2.5 × 10(7) total nucleated cells (TNCs)/kg and a 27.9% probability for white adults. With a cell dose of ≥1 × 10(7) TNCs/kg, the corresponding projected probabilities are 85.6% and 82.1%. The projected probabilities are lower for ethnic minorities. Impetus for using CB HCT was provided by a transplantation of an adult with acute myelogenous leukemia who was not HIV infected. The HCT was performed with a CCR5-Δ32/Δ32 CB unit, and posttransplantation in vitro studies indicated that the patient's peripheral blood mononuclear cells were resistant to HIV infection.

Cell recovery comparison between plasma depletion/reduction- and red cell reduction-processing of umbilical cord blood.

BACKGROUND AIMS: Limited cell dose has hampered the use of cord blood transplantation (CBT) in adults. One method of minimizing nucleated cell loss in cord blood (CB) processing is to deplete or reduce plasma but not red blood cells - plasma depletion/reduction (PDR). METHODS: The nucleated cell loss of PDR was studied, and determined to be less than 0.1% in the discarded supernatant plasma fraction in validation experiments. After testing and archival sampling, the median nucleated cell recovery for PDR processing was 90%, and median CD34(+) cell recovery 88%. In a CB bank inventory of 12 339 products with both pre- and post-processing total nucleated cells (TNC), PDR processing resulted in median post-processing TNC recoveries of 90.0% after testing and archival samples removal. Using the same 10 CB units divided into two halves, we compared directly the recovery of PDR against hydroxyethyl starch red cell reduction (RCR) for TNC, CD34(+) cells and colony-forming units (CFU-GM, CFU-E, CFU-GEMM and total CFU) after parallel processing. We also compared the loss of very small embryonic-like stem cells (VSEL). RESULTS: We demonstrated significantly higher recoveries using PDR for TNC (124%), CD34(+) cells (121%), CFU-GM (225%), CFU-GEMM (201%), total CFU (186%) and VSEL (187%). The proportion of high TNC products was compared between 10 912 PDR and 38 819 RCR CB products and found to be 200% higher for products that had TNC ≥150 × 10(7) (P = 0.0001) for the PDR inventory. CONCLUSIONS: Our data indicate that PDR processing of CB provides a significantly more efficient usage of this valuable and scarce resource.